Hi,
I am currently attempting to express a small (100aa) protein in the pGEX-6P2
vector in BL21 Codon + bugs. I have sequenced the plasmid and there are no
mutations, and the protein is in frame.
I have had successful (GST-fusion) induction of my protein (with protease
inhibitors) over a 3 hour period, at temperatures from room temp, to 30 C,
to 37 degrees C, each time with the same results on a 12% protein resolving
gel. The protein does occur in the soluble fraction.
When I induce the protein it runs 3-5 KDa higher than expected.
My problem however, is that when I induce, not only do I get my protein, but
also a large amount of GST alone being induced. Thus when I attempt to put
the protein mix on the beads, GST appears to outcompete the protein and I
end up with purified GST with no protein at all.
I have no antibody for this protein, and have tried to purify it a number of
times.
Has anyone seen this before, or does anyone have any ideas?
Thanks a lot,
Esme.
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